16-methylene steroids



United StatesPatent O 16-METHYLENE STEROIDS Karl-Heinz Bork, Griesheim,near Darmstadt, and Klaus Briickner, Heinz-.Iiirgen Mannhardt, HaraldMetz, and Fritz von Werder, Darmstadt, Germany, assignorsto E. MerckAktiengeseilschaft, Darmstadt, Germany No Drawing. Filed Sept. 19, 1960,Ser. No. 56,703 Claims priority, application Germany Sept. 23, 1959 I 4Claims. (CI. 260-39145) The present invention relates to a novel seriesof 16- methyiene steroids which possess a good infia'mation arrestingaction which, for example, far exceeds that of hydrocortisone. Thecompounds according to the invention are of the following generalformula I in which R =a -H, 5 OH or =0; X or F, and the 1,2 position cancontain a further double bond.

These steroids can be produced from 16-methylene-17a-hydroxy-progesterone.

According to the invention 16-methylene-17a-hydroxyprogesterone (I) orits l-dehydro derivative (II) is treated with a microorganism effectingll-hydroxylation, whereby 16-methylene 11,170: dihydroXy-progesterone(III) or respectively its l-dehydro derivative (IV) is formed. Insofaras Compound I is used as the starting material it can be subjected toagents affecting 1,2"dehydrogenation either before or after thehydroxylation to obtain the corresponding l-dehydro Compounds II or IY.To prepare the corresponding 9044111010 compound, the16-methylene-11,17a-dihydroxy-progesterone' (III) or its i-dehydroderivative IV is treated with a conventional dehydrating agent and then,if desired, afteresterification of the 17a-hydrox'y group, sequentiallywith hypochlorous or hypobromous acid, a hydrogen halide cleaving agentand hydrogen fluoride. The17-acy1oxy group can be hydrolysed in anydesired reaction step. In addition a 1,2 double bond may be introducedin any desired reaction step with the aid of a conventional 1,2dehydrogenating agent. In the corresponding intermediate or end productsthe ll-hydroxy group can be converted into a keto group with a knownoxidation procedure. Compounds of the above general formula are thusproduced;

A preferred embodiment of the process according to the invention isrepresented by the followingreaction scheme: 0

l Hfl CH1 00 .10 I I -OH A on, Ton,

Patented Nov. 13, 1962 A 2.. iii...

Z1 O we Q I A ii; ii. n IQ (If ll? 4% 3 0... fifi figfi il. $2.. ll/' if'0: so a OH; CHI be be "--on i --on nofli Ton, lac-p: Ton,

The microbiological hydroxylation of Compounds I or II to the ll-hydroxysteroids III or IV can be carried out with the usual microorganisms forthis purpose, such as microorganisms of the Curvularia, Mucor,Streptomyces, Aspergillus, Fusarium, Pencillium, Rhizopus and Bacillusgenera. r

The compounds which are saturated in the 1,2 position can be treated inany desired reaction step with a conventional dehydrogenating agent tointroduce a 1,2 double bond. The dehydrogenation can be effectedchemically or microbiologically. For example, 2,3-dichloro-5, 6-dicyan-p-benzoquinone is suited as a chemical dehydrogenating agent. Thedehydrogenation advantageous is carried out in the presence of asuitable solvent such as dioxane, benzene, ethyl acetate, t-butanol andthe like. The reaction periods lie between 4'and hours during which thereaction mixture is heated under refiux.

All usual microorganisms can be employed for the introduction of the 1,2double bond microbiologically.

Bacillus sphaericus var. fusiformis and Corynebacterium simplex areespecially suited'therefor. Expediently temperatures of 24 to 40 C. areemployed. The 1,2 dehydrogenation products can be extracted from thefermentation medium with chloroform. The substances are isolated incrystalline form by conventional processing of the chloroform extract.

The ll-hydroxy steroids III or IV can be converted into thecorresponding 9,11 unsaturated derivatives V or .VI by treatment with adehydrogenating agent. Depending upon whether an or llfi-hydroxy steroidis used as the starting material one uses one of the usual cisortrans-dehydrogenation methods. In the cis-dehydrogenation one can, forexample, esterify the lla-hydroxy group and subsequently split off thecorresponding acid thermally or with basic agent's. When an llfl-hydroxysteroid is used as the starting material a Smooth transdehydrogenationis effected with phosphorous oxychloride or thionylchloride in pyridine.

Expediently the 17-hydroxy group of the thus obtainedl6-rnethylene-9,1l-dehydrosteroids V or VI is esterified before thehypochlorous or hypobromous acid is added onto the 9,11 double bondthereof. In this way, side reactions, such as an addition of thehypohalous acid on the exocyclic IG-methylene, group, can be avoided.The acylation is effected under usual conditions. Preferably a loweralkonoic carboxylic acid or derivatives thereof suited foresterific-ation are employed for the acylation. Especially good yieldscan be obtained with acetic acid anhydride and acetic acid in thepresence of small quantities of p-toluene sulfonic acid. The thusobtained 16- methylene-17a-acyloxy-9,1l-dehydro-progesterone VII or its1-dehydro derivative VIII can be converted to the corresponding 9a-bromoor 9a-chl0ro1lfi-hydroxysteroid IX or X by treatment with hypobromous orhypochlorous acid or substances yielding such acids, such asNbromoacetamide, N-bromosuccinimide, N-chloroacctamide orN-chlorosuccinimide. Expediently, a small quantity of a strong acid,preferably, perchloric acid, is added in this reaction. If an excess ofhypohalous acid is employed the 9u-halogen-llp-hydroxy-steroidsinitially formed are converted into the corresponding Slot-halogen.-ll-keto compounds.

The 9a-halogen-1lfi-hydroxy-steroids IX and X can be converted into thecorresponding 9,1lB-oxido-steroids XI or XII by treatment with alkalinereagents accordipg to methods known per se.

The cleavage of the 9,8,115-oxido-steroids XI or XII to form theQa-flHOI'O-l lp-hydroxy-steroids XIII or XIV can be efiected bytreatment with hydrogen fluoride, preferably at low temperatures as wellas in the presence of a suitable solvent, such as tetrahydrofurane,chloroform or another chlorinated hydrocarbon.

The saponification of the l7a-acyloxy group can he eifected before orafter the cleavage of the 9,8,11fi-epoxidc with hydrogen fluoride. Theusual alkaline substances used for saponifications of this type, such assodium hydroxide or sodium carbonate, can be the saponification agent.

The ll-hydroxy-steroids obtained according to the invention can beconverted .into thecorresponding l'l-ketosteroids by a usual treatmentwith a mild oxidizing agent. For example, a mixture of chronic acidanhydride and pyridine, chromosulfuric acid in acetone or hypohalousacid is suited as the mild oxidizing agent.

All components shown in the reaction scheme have not been describedpreviously in the literature.

The 16-methylene-17u-hydroxy-progesterone I required as the startingmaterial can, for example, be prepared from16-methyl-pregnadienolone-acetate. This compound can be converted to16u,17a-oxido-16fi-methyl-4- pregnene-3,20-dione by treatment withhydrogen peroxide in an alkaline medium and a following oxidationaccording to Oppenauer. The 16-methylene-l7a-hydroxyprogesterone can beobtained therefrom by splitting the The following examples will serve toillustrate the invention.

EXAMPLE 1 (a) JLB-Hydroxylation of I liters of a nutrient solution of 5%of malt extract, 1% saccharose, 0.2% sodium nitrate, 0.1% dipotassiumphosphate, 0.05% magnesium sulfate, 0.05% potassium chloride and 0.005%iron (II) sulfate with its pH adjusted to 7 Were inoculated with 800 cc.of a shaking culture of Curvularia lunata (Wakker) boadijn in a smallfermenter and incubated at 28 C. with strong aeration and stirring.After 24 hours growth, 5.1 g. of16-methylene-4-pregnene-17a-ol-3,20-dione (I) in 40 cc. of dimethylformamide were added. When the conversion was completed the culture wasexhaustively extracted with chloroform. The extracts were concentratedand passed through a column of activated silica gel. The pure 16-methylene-4-pregnene-11B,17a-diol-3,20-dione (III, 115- OH) was obtainedfrom the middle chromatographic fractions. Melting point: 216-217 C., A241mn;

{1 454; (a) +42 (chloroform) (12) 1,2 Dehydrogenation of III 15 litersof a nutrient solution of a 1% yeast extract (pH 6.8) were inoculatedwith 0.5 liter of a shaking culture of Bacillus sphaericus (collectionE. Merck No. 1001) in a small fermenter. After about 11 hours 7.5 g. of16-methylene-4-pregnene-11 3,17a-diol-3,20 dione (III) in 280 cc. ofmethol were added with constant stirring and aeration. Thedehydrogenation which was followed by paper chromatography was completedin 28 to 33 hours. The fermentation liquor was shaken out three timeswith chloroform and the united extracts concentrated, whereby the16-methylene-1,4-pregnadiene-l1B, 17a-diol-3,20-dione crystallized out.Melting point: 238- 241 C., (a) -37.5 (chloroform), k 243.5m;l,

EXAMPLE 2 (a) Microbiological 11 a-Hydroxylation of l 15 liters of anutrient solution of 5% glucose, 0.1% yeast extract, 0.05% soya beanmeal, 0.3% sodium nitrate, 0.5% magnesium sulfate, 0.001% iron IIsulfate, 1/ 30 m. phosphate buffer according to Siirensen (pH 5.6) wereinoculated with 300 cc. of a shaking culture of Fusarium sp. (collectionE. Merck No. 2083) in a small fermenter and incubated at 28 C. withstrong aeration and stirring. After 24 hours growth, 5 g. of16-methylene-4-pregnene-17a-o1-3,20-dione (I) in 40 cc. of dimethylformamide were added. After 48 hours further incubation under the sameconditions the culture was shaken out three times with chloroform. Theunited extracts were concentrated. The 16-methylene-4-pregnene-11u,17a-diol-3,20-dione (III, Ila-0H) crystallized from the residue andwas recrystallized from acetone. Melting point: 208-210 C., A 241m i,

iZ m. 2, (ab-84 (choroform) (b) Dehydration of III 9.2 g. ofl6-methylene-4-pregnene-11u,17u-diol-3,20- dione (Ila-OH) were dissolvedin 40 cc. of chloroform and 55 cc. of pyridine and 11.2 g. of p-toluenesulfonic acid chloride added while shaking and cooling with ice. Thereaction mixture was allowed to stand overnight and was then poured intowater, extracted with chloroform and processed in the usual manner. Thell-tosylate crys- 6 tallized out of the chloroform solution and wasrecrystallized from methanol. Melting point: 162 C., (a) 2 (chloroform),A 229.5m;i,

1% I. am.

11 g. of the tosylate were heated under reflux in cc. of glacial aceticacid together with 10.3 g. of water free sodium acetate. The reactionmixture was poured into water and the16-methylene-4,9(11)-pregnadiene-17a-o1- 3,20-dione (V) which separatedout filtered off under suction and recrystallized from ethyl acetate.Melting point: 227 C.; (co -23 (chloroform); A 238mp;

ttasao (c) Acetylation of V 40 cc. of acetic acid anhydride and 2 g. ofp-toluene sulfonic acid were added to a solution of 20 g. of 16-methylene-4,9( 11)-pregnadiene-17a-o1-3,20-dione (V) in 200 cc. ofglacial acetic acid. The mixture was allowed to stand 18 hours at roomtemperature and was then slowly poured into water while stirringvigorously. The crude 16-methylene-17a-acetoxy-4,9( 1 1 )-pregnadiene-3,20-dione (VII) separated out in the form of crystals and was filteredoff under suction and purified by recrystallization from anether-acetone mixture. Melting point: 232- 235 C., (a) 64.0 (dioxane), k238.5m,u,

. 3311465 (d) Addition of Hypobromous Acid alt VII 5 g. of16-methylene-17a-acetoxy-4,9(11)-pregnadiene- 3,20-dione (VII) weredissolved in cc. of dioxane and 30 cc. of water. 2.9 g. ofN-bromosuccinimide and 1.1 cc. of 70% perchloric acid were added to themixture and the mixture allowed to stand at room temperature for 30minutes. The mixture was then poured into water with stirring and theprecipitate filtered off under suction. The crude16-methylene-9a-bromo-4-pregnene-1 1,9,17a-dio1-3, 20-dione-17 -acetate(IX) was processed further without purification.

(e). Production of the 9,11 Expovcide XI The crude16-methylene-9a-bromo-4-pregnene-1113,170:- diol-3,20-dione-17-acetate(IX) was dissolved in alcohol. The solution was boiled under reflux for2 hours after the addition of 12 g. of dry potassium acetate. Themixture was poured into water and the resulting emulsion shaken outseveral times with chloroform. The chloroform solution was dried andconcentrated by evaporation. The resulting crude16-methylene-9fi,1lfi-oxido-4-pregnene- 17u-o1-3,20-dione-17-acetate(XI) was purified by recrystallization from ether. Melting point: 194196C., (a) 142.6 (dioxane), A 242mn,

(f) Saponification of XI Eltmsss (g) Cleavage of XIII With HF 6.8 g. of16-methylene-9,8,11B-oxido-4-pregnene-17uo1-3,20-dione (XIII) weredissolved in 68 cc. of absolute chloroform and cooled to 60 C. 41.4 cc.of a mixture of 40 cc. of tetrahydrofurane, 15 cc. chloroform and 36 g.HF were added to the solution and the mixture allowed to stand for 4hours at 30 C. and then poured into aqueous sodium bicarbonate. Themixture was extracted several times with chloroform and the unitedextracts dried and concentrated by evaporation. The residue consistingof crude 9ot-flu0ro-16-methylene-4-pregnene-11p, 17a-diol-3,20-dione(XV) was purified'by recrystallization from ether. Melting point:247-250 C., +526 (ethanol), A 2385mm (M) Microbiological Dehydrogenationof XV Analogously to Example lb, 7.8 g. of 9a-fluoro-16-methylene-4-pregnene-1 1fi,17m-diol-3,20-dione (XV) were dehydrogenatedto 9a-fluoro-16-methylene-1,4-pregnadiene-llfl,l7a-diol-3,20-di0ne (XVI)with Bacillus sphaericask Melting point: 271-275? C., ((1);, +300(ethanol), A 2385mm i'im. 3

(I1 Chemical Dehydrogen-crtion of XV EXAMPLE 3 (q) Microbiological andChemical Dehydrogenation of l Analogously to 2h and 211 8.0 g. of16-methylene- 4 -pregnene-17a-ol3,20-dione (I) were dehydrogenated toI6-n1ethylene-1,4 pregnadiene-17a-ol-3,20-dione (II).

(b) Microbiological 11,8 Hydroxylation of II Analogously to Example 1a,the 16-methylene-1,4- pregnadiene-l1,6,I7a-diol-3,20-dione (IV, llfi-OH)was prepared from 16-methy1ene-1,4-pregnadiene-1711-01-330- dione (II).The product was identical with that of Example 1b.

8 EXAMPLE 4 (a) Oxidation of III 2.3 g. of16-methylene-4-pregnene-lla,17a-diol-3,20- dione (III, Hot-OH) weredissolved in 23 cc. of absolute pyridine and a mixture of 2.3 g. ofchromic acid anhydride' and 23 cc. of pyridine added thereto at 0 C.After 12 hours the reaction mixture was poured into 250 cc. of ethylacetate, the precipitate filtered ofi under suction and washedthoroughly with ethyl acetate. The combined ethyl acetate solutions wereconcentrated whereby the 16- methylene-4-pregnene-17u-ol-3, ll,20-trione (XIX) crystallized out. k 23 8m,u..

(b) Oxidation of XV 4.2 g. of 9a-fluoro16-methylene-4-pregnene-1118,17wdiol-3,20-dione (XV) were dissolved in cc. of absolute pyridine andmixed with a mixture of 4.2 g. of chromic acid anhydride and 42 cc. ofpyridine at 0 C. After 12 hours the reaction mixture was poured into 500cc. of ethyl acetate, the precipitate filtered off and washed thoroughlywith ethyl acetate. The combined ethyl acetate solutions wereconcentrated whereby the 9e-fiuoro-16-methylene4-pregnene-17a-ol-3,11,20-trione (XVII) crystallized out.

(c) Oxidation of X VI Analogously to Example 415, the9a-fiuoro-16-methylene-l,4-pregnadiene-17u-ol-3,11,20-trione (XVIII) wasprepared from 9a-fiuoro-16-methylene-1,4-pregnadiene-1lfi,17oz-diOl-3,20-Cli0ne (XVI).

We claim:

1. 9a-fluoro-16-methylene 4 pregnene-ll ,6,17e-diol- 3,20-dione.

V 2. 9a-fluoro-16-methylene 1,4 pregnadiene-llfiJhdiol-3,20-dione.

3. 16-methylene-4-pregnene-17u-ol-3 ,1 1 ,20-trione.

4. 16-methy1ene-1,4-pregnadiene-17a-ol-3,11,20-trione.

References Cited in the file of this patent UNITED STATES PATENTSAgnello et al. Dec. 23, 1958 Agnello et al. Dec. 1, 1959

